Integrin signaling is critical for chondrogenesis, and its alteration is a causal factor in the development of skeletal diseases such as osteoarthritis. This project takes advantage of exciting new findings on Kindlin-2, a key regulator of integrin signaling, and seeks to define its role in control of chondrogenic cell behavior. Studies by the applicants have shown that ablation of Kindlin-2 in chondrogenic cells results in severe defects in chondrogenesis in mice. Furthermore, loss of Kindlin-2 impairs TGF-?1 signaling and the expression of Sox9, a master regulator of chondrogenesis. Additionally, there is evidence that Kindlin-2 localizes not only to focal adhesions (FAs) but also to the nuclei of chondrocytes. Based on these and other studies, the applicants hypothesize that Kindlin-2 works in concert with its cytoplasmic and nuclear binding partners to regulate TGF-?1 signaling and Sox9 expression and thereby control chondrocyte functions and chondrogenesis. To test this, the applicants propose the following three aims. Aim 1 is to determine the mechanisms by which Kindlin-2 regulates Sox9 expression in chondrocytes. The applicants will employ a combination of molecular, cellular and genetic strategies to test the hypothesis that Kindlin-2 regulates Sox9 expression through not only its interactions at FAs but also its interactions in the nuclei. Aim 2 s to determine the roles and mechanisms of Kindlin-2 in TGF-?1 activation and signaling. The applicants will test the hypothesis that Kindlin-2 regulates TGF-?1 activation and downstream signaling by interacting with key components of the TGF-?1 signaling pathway. Aim 3 is to determine the functions of the Kindlin-2-Sox9 signaling axis in chondrogenesis in vivo. The applicants will employ conditional knockout and transgenic mouse models to define the functional contribution of the Kindlin-2-Sox9 signaling axis to chondrogenesis in vivo. These studies will shed light on the interplay between three key control elements in chondrogenesis (integrin signaling, TGF-?1 signaling and Sox9 expression) and the underlying mechanisms, which will significantly improve our understanding of the fundamental mechanisms governing chondrocyte behavior and may lead to development of novel strategies for alleviating common skeletal diseases such as osteoarthritis.